Share

DNA Sequencing

ITS Region: PCR for species verification

Master Mix for a 20 µl reaction - Place the chemicals below in a tube in the following order, keeping the chemicals on ice while you are working:

2 µl 10X PCR buffer (standard buffer with New England Biologicals or ProMega Taq)

0.5 µl dNTPs (10 mM, ProMega or New England Biologicals)

1 µl 10X ITS-1 primer (5 µM; TCC GTA GGT GAA CCT GCG G)

1 µl 10X ITS-4 primer (5 µM; TCC TCC GCT TAT TGA TAT GC)

14.4 µl PCR water

0.1 µl Taq polymerase

Total = 19 µl

Add 1 µl  DNA from sample (10-25 ng DNA/ µl)

OR... Master Mix for a 25 µl reaction - in the following order…everything kept on ice:

5 µl 10X PCR buffer (standard buffer with New England Biologicals)

1 µl dNTPs (10 mM, ProMega or New England Biologicals)

1 µl 10X ITS-1 primer (5 µM; TCC GTA GGT GAA CCT GCG G)

1 µl 10X ITS-4 primer (5 µM; TCC TCC GCT TAT TGA TAT GC)

15.75 µl PCR water

0.25 µl Taq polymerase

Total = 24 µl

Add 1 µl DNA from sample (18-25 ng DNA/ µl)

Thermocycler program for ITS region PCR:

Step 1:   94°C     5 minutes

Step 2:   94°C     1 minute

Step 3:   55°C     1 minute

Step 4:   72°C     1 minute

Step 5:   Go to step 2, 34X

Step 6:   72°C     10 minutes

Step 7:   4°C       Hold indefinitely

coxII  region: PCR for species verification

Master Mix for a 50 µl reaction - in the following order…everything kept on ice:

5 µl 10X PCR buffer (standard buffer with Biolab)

1 µl dNTPs (Biolab)

1 µl 10X FM58 primer (5 µM; CCA CAA ATT TCA CTA CAT TGA)

1 µl 10X FM66 primer (5 µM; TAG GAT TTC AAG ATC CTG C)

1.5 µl 50 mM MgCl2 (Invitrogen)

0.1 µl Taq polymerase (1.25U, Biolab)

39.4 µl PCR water

Total = 48 µl

2.0 µl DNA from sample (20-30 ng DNA/µl)

Run the thermocycler as follows for 35 cycles:

Step 1:   94C      2 minutes

Step 2:   94C      1 minute

Step 3:   56C      1 minute

Step 4:   72C      2 minute

Step 5:   Go to step 2, 34X

Step 6:   72C      10 minutes

Step 7:   4C        Hold indefinitely

Direct PCR = An alternative to extracting DNA for sequencing

Grow the isolate on an agar (probably PDA) where it will grow abundantly on the agar surface.

Make the Master Mix as noted below and dispense it into a microcentrifuge tube that will be put into the thermocycler.

Using a sterile disposable pipet tip (one of those used to dispense µl amounts) attached to a pipetor, scrape the tip across the colony 3-4 times, 1-2 cm per scrape, but try to not pick up any agar on the tip. The objective is to break some of the hyphae and 'contaminate' the tip with some DNA from the hyphae.

Plunge the tip into the master mix in the microcentrifuge tube. Swirl it around and also take up some of the master mix and expel it back into the tube a few times. The objective is to put the DNA into the master mix.

Put the tube in the thermocycler as noted above.

Moorman and his students have done this successfully for the ITS PCR. It does not work for every isolate.

He has not tried it for coxII but it should work.

ITS Region - Direct PCR Master Mix

Master Mix for a 20 µl reaction -  Place the chemicals below in a tube in the following order, keeping the chemicals on ice while you are working:

2 µl 10X PCR buffer (standard buffer with New England Biologicals or ProMega Taq)

0.5 µl dNTPs (10 mM, ProMega or New England Biologicals)

1 µl 10X ITS-1 primer (5 µM; TCC GTA GGT GAA CCT GCG G)

1 µl 10X ITS-4 primer (5 µM; TCC TCC GCT TAT TGA TAT GC)

15.4 µl PCR water

0.1 µl Taq polymerase

Total = 20 µl

OR... Master Mix for a 25 µl reaction -  in the following order…everything kept on ice:

5 µl 10X PCR buffer (standard buffer with New England Biologicals)

1 µl dNTPs (10 mM, ProMega or New England Biologicals)

1 µl 10X ITS-1 primer (5 µM; TCC GTA GGT GAA CCT GCG G)

1 µl 10X ITS-4 primer (5 µM; TCC TCC GCT TAT TGA TAT GC)

16.75 µl PCR water

0.25 µl Taq polymerase

Total = 25 µl

Regardless of which PCR you run, check to be certain a product was obtained:

Run 3-5 µl of PCR reaction product on 1.5% agarose gel electrophoresis with 2 µl of loading dye (see below).

Moorman's lab has ceased using the very toxic ethidium bromide for visualizing DNA and now uses Amresco EZ-Vision Three® (DNA dye/loading buffer; Amresco, 30175 Solon Inc. Pkwy, Solon, OH 44139; 1-800-448-4442)